Glycoprotein mediated membrane fusion is a common mechanism for entry of enveloped viruses. The glycoprotein on the viral surface is usually present in a metastable conformation that undergoes a dramatic refolding during the fusion process. Inhibition of such fusion by small molecule compounds is a unique approach to delineate the mechanism of glycoprotein mediated membrane fusion, and to potentially search for novel antiviral drugs. In our preliminary studies, a class of compounds was found to inhibit membrane fusion mediated by hemagglutinin (HA) of influenza viruses at picomolar concentrations. To further study the mechanism of inhibition of HA-mediated membrane fusion, three aims are presented in this proposal: Aim 1. Structural studies. Three experiments in preliminary studies suggested that the compounds target HA for their inhibitory activities. In this aim, cocrystallization of HA will be carried out with a panel of inhibitors and their structures will be solved. The crystal structures will show the exact binding site for these compounds and may reveal what structural changes may have been caused by these compounds. Aim 2. in vitro fusion studies. HA mediated fusion of liposomes will be studied in the presence of inhibitors. Steps of fusion inhibition by the inhibitors will be defined. Further more, recombinant HA will be treated with different proteases in order to map the conformationally sensitive regions. The inhibitors will be included in the proteolysis study to define on which region the inhibitors have the most impact. Aim 3. Mutational studies. Another approach to understand the mechanism of inhibition by this class of compounds is to select resistant mutants. Studies on the resistant mutants will help us to understand how HA changes conformation to mediate membrane fusion. Another type of resistant mutants could be those that alter the binding site for inhibitors. Such mutants were not identified previously because of lack of knowledge on inhibitor binding sites. The result from aim 1 will help us in this aspect. We will also design site-specific mutants based on the outcome of Aim 1. Aim 4. Chemical synthesis. Expanded libraries derived from the lead compounds have been synthesized. These compounds contain a similar overall structure, but with very different affinities. Further modification of the compounds may serve two purposes. First, new compounds may cause different structural changes due to variations in substituents. If we solve a series of structures that have structural changes from small to large as caused by different inhibitors, we can model the pathway of structural changes that trigger the conformational change required for membrane fusion. Second, we can test if our understanding of the conformational change in HA is correct or not by designing new inhibitors.